Non-tuberculous Mycobacterium spp. are ubiquitous in the environment and cause infections and pseudo-infections in health care settings throughout the world. Among these, the rapidly-growing Mycobacterium (RGM) species, M. abscessus (subsp. abscessus and subsp. massiliense), M. chelonae, and M. fortuitum are particularly problematic due to their ubiquitous presence in hospitals'water sources and the difficulty of treating the infections they cause. There is increasing awareness of M. abscessus subspp. abscessus and massiliense in particular as emerging pathogens. Of particular concern is the increasing frequency with which glutaraldehyde-resistant RGM are being associated with nosocomial outbreaks, sometime reaching epidemic proportions as recently documented in Brazil. Glutaraldehyde (GTA) is the most widely used chemical disinfectant for heat-sensitive medical devices in hospitals worldwide. What is known of the mode of action of this disinfectant in other bacteria suggests that changes in the surface-exposed composition of the cel envelope resulting in decreased binding and/or penetration of GTA may be one of the mechanisms through which RGM develop high levels of resistance. Because of the important role played by the mycobacterial outer membrane in drug susceptibility and host-pathogen interactions, there is thus some concern that the widespread use of GTA and related aldehyde disinfectants in clinical settings is impacting on the selection of resistant populations of RGM with possible consequences on cross-resistance to drugs and pathogenicity. In support of these assumptions, we recently demonstrated that reduced porin expression was one of the mechanisms through which M. smegmatis and M. chelonae develop high levels of resistance to GTA and a further aldehyde disinfectant, ortho-phthalaldehyde (OPA). In part due to defects in porin expression, the GTA/OPA-resistant M. chelonae isolate under study also displayed unusually high levels of resistance to a number of drugs including rifampicin, ciprofloxacin, clarithromycin, erythromycin, vancomycin, tetracycline and linezolid. Further, our preliminary results indicate that, compared to a GTA-susceptible reference M. massiliense clinical isolate, the epidemic M. massiliense isolates from Brazil display unusual resistance to aminoglycosides and much increased pathogenicity in mice. We have assembled a collection of GTA-resistant isolates of M. chelonae and M. abscessus subsp. massiliense responsible for nosocomial infections and pseudo-infections in different regions of the world. The fact that not all of these isolates display cross-resistance to OPA (unlike the M. chelonae isolate described above) indicates that there must exist more than one mechanism of resistance to aldehyde disinfectants in mycobacteria. We propose to use our collection of clinical isolates together with a combination of genomic, transcriptomic, genetic and biochemical approaches to investigate the molecular mechanisms of resistance of RGM to aldehyde-based disinfectants and to study the potential impact the phenotypic changes undergone by these strains might have had on their susceptibility to drugs and pathogenicity. The results of this study could lead to the implementation of optimized strategies for the disinfection of medical devices and suggest more efficient treatments to cure those nosocomial infections caused by disinfectant-resistant isolates. PUBLIC HEALTH RELEVANCE: We propose to elucidate the mechanisms of resistance of nosocomial rapidly-growing mycobacterial pathogens to aldehyde disinfectants and to study the impact of the phenotypic changes undergone by resistant isolates on their susceptibility to drugs and pathogenicity.